. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.Coronaviruses are positive-sense RNA enveloped viruses that belong to the Coronaviridae family in the Nidovirales order. These viruses are found in a wide variety of animals and can cause respiratory and enteric disorders of diverse severity (11,18). In the past 5 years, several human and animal coronaviruses have been discovered, including the highly pathogenic virus responsible for the severe acute respiratory syndrome (SARS-CoV) (34,58, 60,64,68,69). Coronavirus particles consist of a helical nucleocapsid structure, formed by the association between nucleocapsid (N) phosphoproteins and the viral genomic RNA, which is surrounded by a lipid bilayer where three or four types of structural proteins are inserted: the spike (S), the membrane (M), and the envelope (E) proteins and, for some coronaviruses only, the hemagglutinin-esterase (HE) protein (for a review, see reference 14). Once sufficient amounts of new genomic RNA and structural proteins have been produced, assembly of particles occurs. Assembly and release of virions are the last stages of the virus life cycle.The triple-spanning membrane glycoprotein M drives the assembly of coronavirus, which bud into the lumen of the endoplasmic reticulum-Golgi intermediary compartment (ERGIC) (32,33,62,63). M is the most abundant envelope protein that sorts viral components to be incorporated into virions. M oligomerization, mainly driven by its transmembrane domain, is believed to allow the formation of a lattice of M proteins at ERGIC membranes (16, 41). S and E membrane proteins are integrated into the lattice through lateral interactions with M, whereas N and viral RNA interact with M C-terminal domain, which is exposed to the cytosol (4,8,15,19,30,36,48,54,55). The coronavirus S protein, responsible for receptor binding and membrane fusion, does not seem to have any major role in coronavirus assembly. Recent studies show that E is a viroporin that forms ion channels (46,66,67). Despite its minor incorporation into virion particles (7,22,40), the small E protein plays an important but not fully understood role in virus morphogenesis and budding (20,35,70). Studies performed on coronaviruses, including the SARS-CoV, demonstrate that depletion of the E gene from coronavirus genome strongly diminish virus growth and particle formation (9,12,35,37,57). The N protein self-associates and encapsidates the RNA genome for incorporation i...

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Coronavirus tropism is predominantly determined by the interaction between coronavirus spikes and the host receptors. In this regard, coronaviruses have evolved a complicated receptor-recognition system through their spike proteins. Spikes from highly related coronaviruses can recognize distinct receptors, whereas spikes of distant coronaviruses can employ the same cell-surface molecule for entry. Moreover, coronavirus spikes can recognize a broad range of cell-surface molecules in addition to the receptors and thereby can augment coronavirus attachment or entry. The receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) is dipeptidyl peptidase 4 (DPP4). In this study, we identified membrane-associated 78-kDa glucose-regulated protein (GRP78) as an additional binding target of the MERS-CoV spike. Further analyses indicated that GRP78 could not independently render nonpermissive cells susceptible to MERS-CoV infection but could facilitate MERS-CoV entry into permissive cells by augmenting virus attachment. More importantly, by exploring potential interactions between GRP78 and spikes of other coronaviruses, we discovered that the highly conserved human GRP78 could interact with the spike protein of bat coronavirus HKU9 (bCoV-HKU9) and facilitate its attachment to the host cell surface. Taken together, our study has identified GRP78 as a host factor that can interact with the spike proteins of two , the lineage C MERS-CoV and the lineage D bCoV-HKU9. The capacity of GRP78 to facilitate surface attachment of both a human coronavirus and a phylogenetically related bat coronavirus exemplifies the need for continuous surveillance of the evolution of animal coronaviruses to monitor their potential for human adaptations.

Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses with genome sizes of approximately 30 kb. They belong to the family Coronaviridae in the order Nidovirales and are currently classified into four major genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus (1). Coronaviruses can infect a wide range of mammals, as well as birds (2). The broad species tropism is predominantly attributed to the high diversity in receptor usage across different coronaviruses. To date, six coronaviruses are known to infect humans, and they utilize different surface molecules for cell entry. In particular, human coronavirus 229E (HCoV-229E) binds aminopeptidase N (APN) (3), and human coronavirus OC43 (HCoV-OC43) binds O-acetylated sialic acid (4). Severe acute respiratory syndrome coronavirus (SARS-CoV) (5) and human coronavirus NL63 (HCoV-NL63) (6) both bind angiotensin I converting enzyme 2 (ACE2). The receptor for human coronavirus HKU1 (HCoV-HKU1) has not been defined. However, O-acetylated sialic acid has been suggested as an attachment factor that contributes to the binding of HCoV-HKU1 to the cell surface (7). Middle East respiratory syndrome coronavirus (MERS-CoV) is the sixth coronavirus known to cause infection in humans (8). Intriguingly, MERS-CoV utilizes a unique cellular receptor, dipeptidyl peptidase 4 (DPP4), for virus entry (9). The host cell receptors for a number of animal coronaviruses have also been identified. For instance, porcine transmissible gastroenteritis coronavirus (TGEV) binds APN (10), and the prototype betacoronavirus mouse hepatitis virus (MHV) uses